T lymphocyte activation, and hence the initiation of an immune response, requires recognition by T cell receptors of complexes consisting of peptide fragments of foreign antigens bound to major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APC). Despite the critical nature of this first step in immune recognition, little is known about the mechanistic aspects of antigen processing, such as how the antigen fragments are produced, and how and where within the APC they encounter MHC molecules. A growing body of evidence suggests that the MHC contains genes which function in the transport of processed antigen into vesicles of the secretory system for binding by MHC class molecules and subsequent vesicular transport to the cell surface. The recent cloning of two MHC genes, called HAM1 and HAM2, which are homologous to a large superfamily of both prokaryotic and eukaryotic transporters represents a major step towards understanding the nature of antigen processing. This proposal deals with the structural and functional characterization of these two genes. Two types of experiments will be done to provide formal proof of the involvement of the HAM1 and HAM2 gene in antigen processing. First, full length, wild type HAM1 and HAM2 cDNAs will be introduced into cell lines carrying MHC-linked mutations that inactivate the antigen processing pathway. Second, mice deficient in HAM1 and HAM2 expression will be produced by introducing defective copies of these genes into embryonic stem cells by homologous recombination, and subsequently injecting these modified cells into mouse blastocysts. In order to further characterize the function of the HAM1 and HAM2 gene products, antibody will be produced to synthetic peptides derived from the deduced amino acid sequences. This antibody will be used immunohistochemically to localize the corresponding proteins within the cell (thus identifying the subcellular compartment where peptide meets MHC molecule), and to aid in their purification. Attempts will be made to reconstitute peptide transport in in vitro systems, and to analyze the specificity of the transport process. In addition to the above, the functional significance of two novel MHC class II-like genes will be addressed by examining their polymorphism and by producing antibody which would allow identification of the corresponding gene products.